Engineering a Pseudomonas putida Chassis for Targeted Enzymatic Litholysis: A Sink-Driven Synthetic Biology Framework for Calcium Oxalate and Cholesterol Calculi Dissolution

Nephrolithiasis and cholelithiasis remain prevalent conditions managed primarily by mechanical or surgical interventions, including extracorporeal shock wave lithotripsy (ESWL) and endoscopic extraction. These approaches, while clinically effective, do not directly address biochemical recurrence and are associated with procedural morbidity. We propose a synthetic biology–inspired, cell-free enzymatic litholysis framework based on Pseudomonas putida KT2440 as a heterologous expression chassis for two enzymatic modules: (i) the oxalyl-CoA decarboxylase/formyl-CoA transferase (OXC/FRC) system from Oxalobacter formigenes for oxalate depletion, and (ii) cholesterol oxidase from Pseudomonas aeruginosa for cholesterol modification. Unlike direct enzymatic surface degradation, we introduce a sink-driven equilibrium model, in which continuous enzymatic depletion of soluble substrates shifts dissolution equilibria at the crystal–fluid interface, enabling gradual mobilization of calcium oxalate and cholesterol-rich calculi. An order-of-magnitude feasibility analysis suggests that the required enzymatic depletion flux falls within the same magnitude as physiological urinary oxalate excretion, supporting physical plausibility. This framework reframes lithiasis treatment as a controlled biochemical depletion system rather than mechanical fragmentation. Keywords: nephrolithiasis; cholelithiasis; synthetic biology; Pseudomonas putida; Oxalobacter formigenes; sink-driven equilibrium; enzymatic litholysis; targeted enzyme therapy.